Persistence of COVID-19 Human Milk Antibodies After Maternal COVID-19 Vaccination: Systematic Review and Meta-Regression Analysis

The World Health Organization (WHO) declared COVID-19 a pandemic. The Centers for Disease Control and Prevention (CDC), WHO, and American College of Obstetricians and Gynecologists (ACOG) recommend vaccination of pregnant and lactating women, aiming to protect both mothers and their infants through transplacental and human milk antibody transmission. This study aims to assess the quantity of antibodies in human milk and determine the effect of time, vaccine type, and dose on antibody level. Single-arm prospective observational studies reporting the COVID-19-specific antibody level in human milk after COVID-19 vaccination during pregnancy or lactation were included. PubMed, Scopus, Cochrane, EBSCO, and Web of Science were searched from December 2019 to November 22, 2022. Data were extracted in a uniform Google sheet. A total of 2657 studies were identified. After the removal of duplicates and screening, 24 studies were included in the systematic review and meta-regression. Human milk COVID-19-specific antibody levels increased with subsequent vaccine doses, as reflected by a positive relationship for the second (coefficient=0.91, P-value 0.043 for IgA and coefficient=1.77, P-value 0.009 for IgG) and third (coefficient=1.23, P-value 0.0029 for IgA and coefficient=3.73, P-value 0.0068 for IgG) doses. The antibody level exhibited a weak positive relationship with the follow-up time (coefficient=0.13, P-value 0.0029 for IgA and coefficient=0.18, P-value 0.016 for IgG). Only one of the 38 infants showed detectable COVID-19 IgM and IgA antibody levels in their blood. There was an increase in the neutralizing activity of COVID-19 antibodies in human milk following the COVID-19 vaccination. From the analysis of published data, we found high positive levels of antibodies in human milk that increased with subsequent doses. Additionally, the human milk antibodies exhibit a positive neutralizing effect. Only one infant had detectable COVID-19 IgM+IgA antibodies in the blood. Further research is needed to discuss infant protection through a mother's vaccination.


Introduction And Background
In December 2019, the authorities in Wuhan, China, officially informed the World Health Organization (WHO) of the existence of numerous cases of a newly identified respiratory disease.China promptly confirmed the existence of a new pathogenic virus belonging to the Coronaviridae family.The complete genome of this coronavirus was made publicly available on January 28, 2020, and it was officially designated as COVID-19 by WHO on February 11 [1].The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, causing a disastrous disease in humans known as coronavirus disease 2019 or COVID-19.This disease has had an enormous impact not only on global health but also on the economy [2].
The COVID-19 pandemic shifted research priorities, and since then, developing reliable and effective vaccines has become a global hope.Administering preventive strategies against infection and finding cures to lessen the symptoms have become a primary research concern [3,4].
As per the established guidelines for clinical trials, COVID-19 vaccines initially did not include trials in pregnant and/or lactating women.However, later on, the Centers for Disease Control and Prevention (CDC), WHO, and the American College of Obstetricians and Gynecologists (ACOG) recommended the vaccination of all eligible people, including pregnant women or those in the postpartum period [8,9].Furthermore, it has been shown that vaccination can promote passive immunization in infants and provide a first line of defense against different pathogens.
Breast milk is the best source of nutrition and immunity through the transmission of antibodies, either following a natural infection or vaccination [10].Breast milk is composed of a wealth of specific immuneprotective factors.Among these, human milk antibodies comprise largely secretory immunoglobulins A (IgA, >90%), as well as secretory immunoglobulins M (IgM, 8%) and immunoglobulins G (IgG, 2%) [11].
Previous systematic reviews have discussed the safety, acceptance, and effectiveness of the COVID-19 vaccine and the existence of COVID-19 antibodies in human breast milk following SARS-CoV-2 vaccination among pregnant and lactating women.The focus primarily revolved around mRNA vaccines [12][13][14][15].Only one meta-analysis was conducted on the presence of COVID-19 antibodies in human milk without discussing the effect of time, type of vaccine, and the number of vaccine doses on the antibody levels [16].
Therefore, the aim of this study was to assess the quantity of COVID-19-specific antibodies in human milk and determine the effect of time, vaccine type, and the number of doses on the COVID-19-specific antibody level.

Review Methods
We followed the Preferred Reporting Items for Systematic Review and Meta-Analysis 2020 (PRISMA) criteria in conducting this systematic review and meta-analysis [17].We prospectively registered our study on the PROSPERO database (CRD42023439229).

Eligibility Criteria and Selection Process
Two independent authors (NH and EB) conducted a comprehensive search on the following databases: Scopus, PubMed, Cochrane, EBSCO, and Web of Science.We used the following search strategy: (SARS-CoV-2 vaccine) OR (COVID-19 vaccine) AND (breast milk) AND (antibody level).We then uploaded all the identified records on the Rayyan website, and duplicates were removed using Rayyan [18].Then, we assessed the remaining studies for eligibility.
We included single-arm prospective observational studies that met the following criteria: (1) their population is breastfeeding women who were vaccinated or decided to be vaccinated against COVID-19 during pregnancy or breastfeeding by any available vaccine and with any number of doses; and (2) studies that measure COVID-19-specific antibody levels (IgA, IgG, or IgM) in milk and their persistence throughout time.The exclusion criteria were case reports, case series, double-arm studies, studies on antibodies against human coronaviruses (including SARS-CoV-2), and those searching for COVID-19 viral particles in human breast milk.We screened the studies in two steps.First, title and abstract screening by Rayyan and then fulltext screening.We also screened the references of all the studies included in the full-text screening.The screening was done by two independent authors (AM and EB), and disagreement was resolved by the third author (NH).

Data Extraction and Quality Assessment
Two independent authors (AM and EB) extracted the data into a uniform Google sheet, and any disagreement was resolved by the third author (NH).The following data were extracted: (1) a summary of included studies, (2) population characteristics of included studies, and (3) primary and secondary outcomes.
The Newcastle-Ottawa scale was used to assess the risk of bias in the included studies by two independent authors (AM and EB), which included the following domains: (1) Selection, (2) Comparability, and (3) Outcome.The study was judged to be of good quality if it got three or four stars in the selection domain and one or two stars in the comparability domain and two or three stars in the outcome domain.The study was considered of fair quality if it deserved two stars in the selection domain and one or two stars in the comparability domain and two or three stars in the outcome domain.The study was reported to be of poor quality if it gets zero or one star in the selection domain or zero star in the comparability domain or zero or one star in the outcome domain [19].

Data Analysis
According to our data, the units of measurement were completely different, and there was no statistical way to convert them to each other.We used log-transformed mean to reduce variance and skewness in the data [20,21].We used a meta-regression model to test the effect of the vaccine type, number of doses, and time on COVID-19-specific antibody levels in human milk using the R software (R Foundation for Statistical Computing, Vienna, Austria) version 1.4.1717,R-4.3.2 for Windows.Different units of measurement do not affect the meta-regression model [22].We assessed the level of heterogeneity using I2 and chi-square test; significant heterogeneity was considered when chi-square P<0.1.Time was measured in weeks.For studies that report time in range, we took the mean of the time range.

Results
A total of 2633 studies were identified through PubMed, Cochrane, Web of Science, Scopus, and EBSCO, which were then uploaded to Rayyan.An additional 25 studies were added from manual screening.After the removal of duplicates by Rayyan, 2106 articles were screened for eligibility.We excluded 2006 studies based on the exclusion criteria during the title and abstract screening, and another 80 studies were excluded in the full-text screening step.We included only single-arm studies.Finally, we included 24 studies in our systematic review, and only 19 were eligible for the meta-analysis.The PRISMA flow diagram is shown in Figure 1 [23].

Characteristics of the Included Studies
All the included studies were longitudinal prospective observational studies, comprising 824 women.In 21 studies, the mothers received the vaccine postpartum.Only three studies included women who received the vaccine during pregnancy.Eighteen studies reported the use of mRNA COVID-19 vaccines, specifically Pfizer or Moderna vaccines, and two of them reported the use of three doses of Pfizer.Four studies reported the use of four available COVID-19 vaccines (Pfizer, Moderna, Janssen, AZ).Previous SARS-CoV-2 infection was reported only in four studies in a range of 3%-13% of participants, while five studies did not report the state of prior infection.There was variability in the timeline of human milk COVID-19-specific antibody measurement that ranged from one month up to nine months from the first dose and only one study up to three months after the Pfizer vaccine booster dose.The summary of studies and patient characteristics is shown in Tables 1, 2.       [26,31,35,36].In the comparability domain, all studies were considered low quality due to the inclusion of single-arm studies.Therefore, it would be unfair to judge these studies according to the comparability domain.In the outcome domain, all studies are considered high quality except for Juncker et al. [29] due to a short follow-up time and inadequate follow-up data (Figure 2) [19].3) (Figures 3, 4).There was a strong positive relationship between the Moderna vaccine and human milk COVID-19-specific antibody (coefficient=6.22,P-value <0.001 for IgA and coefficient=6.56,P-value <0.0001 for IgG).While for J&J, there was a weak positive relationship for IgA (coefficient=0.39,P-value 0.615) and a negative relationship for IgG (coefficient=-0.33,P-value 0.76) (Table 3).Five studies reported the use of any type of vaccine without reporting the results according to the type of vaccine.Therefore, we analyzed these studies separately.Human milk COVID-19-specific antibodies have a weak positive relationship with subsequent vaccine dose (coefficient=0.65,P-value 0.62 for IgA and coefficient=0.3,P-value 0.84 for IgG) and time (coefficient=0.035,P-value 0.28 for IgA and coefficient=0.079,P-value 0.023 for IgG) (Table 4).There was significant heterogeneity among studies regarding all outcomes, with an I2 value exceeding 99% (P-value>0.0001).

Level of Antibody in Infant Blood
Four studies investigated the presence of SARS-CoV-2-specific antibodies in 38 infant serums after mother immunization; only one of the 38 infants showed detectable anti-SARS-CoV-2 IgM+IgA antibodies [26,36,37,43].

Neutralizing Activity of Human Milk SARS-CoV-2 Antibody Following Vaccination
Five studies investigated the neutralizing activity of human milk antibodies post-SARS-CoV-2 vaccination.All these studies showed an increase in the neutralizing activity of human milk post-vaccination compared to pre-vaccination.With time, there was a decline in the neutralizing activity.Perez et al. showed a decrease of inhibition % activity with time to a level < cutoff value at six months post-vaccination.Stafford et al.

FIGURE 1 :
FIGURE 1: The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) flow diagram.

FIGURE 2 :
FIGURE 2: Quality assessment using the Newcastle-Ottawa quality assessment scale

TABLE 2 : Summary of the studies that were not included in the meta-regression analysis.
According to the Newcastle-Ottawa quality assessment scale, in the selection domain, all studies were considered good or fair quality except for Calil et al.,Kelly etal., Rosenberg-Friedman et al., and Perez et al.